Monoclonal antibodies have become major constituents of the burgeoning field of biotechnology related products. It has become apparent that for the development and general distribution of MAb diagnostic and therapeutic products, large scale production techniques will be necessary.
Although MAbs can be produced in vivo by collection of ascitic fluid, in vitro mass cell culture is in many ways preferable (Samoilovich et al., 1987, Hybridoma technology: new developments of practical interest. J. Immunol. Methods 101, 153). Due to the significant production expenses incurred in these large scale cultures, obtaining maximal yields of reactive MAb is indeed important (Velez et al., 1986, Kinetics of monoclonal antibody production in low serum growth medium. J. Immunol. Methods 86, 45); (Reuveny et al., 1986, Comparison of cell propagation methods for their effect on monoclonal antibody yield in fermentors. J. Immunol. Methods 86, 53). To date, most reports in this area describe methods for improving MAb yields by optimizing media formulations and by controlling environmental culture conditions (Samoilovich et al., supra). Lacking, however, are discussions of the selection and maintenance of initial variant hybridoma starter cultures capable of secreting increased MAb concentrations.
The method for producing monoclonal antibodies was first described by Kohler and Milstein in 1975. Since that time, many variations and improvements have been made on their process (for a general review, see Methods in Enzymology, 121, 1986). This invention is not concerned with the process of constructing new hybridomas but rather focuses on the cloning and re-selection of existing hybridoma cell lines.
Hybridoma cells result from the fusion of tetraploid myeloma cells with diploid, antigen stimulated lymphocytes. The myeloma cell partner confers the ability to grow indefinitely in culture while the genetic information which codes for antibody specificity and type emanates from the lymphocyte. The resultant hybridoma cell is capable of growing indefinitely and secreting a uniformly consistent and homogeneous MAb of known specificity. Due to its polyploid nature, the resultant hybridoma is also genetically unstable. Genetic instability of hybridomas is a well documented fact which can, over time, lead to profound phenotypic changes. Quite frequently, hybridoma cultures will decrease the rate at which they secrete MAb and correspondingly increase their growth rate. The combined result of those changes causes the culture gradually to lose its capacity to produce and secrete MAb while consuming ever increasing amounts of substrate which results only in greater cell mass rather than MAb. Isotype switching is another phenotypic change which can occur as a hybridoma cell line is left in continuous culture. Isotype switching occurs when small sub-populations of cells within a culture change over to a different protein backbone (isotype) while maintaining the identical antigen specificity. The most detrimental phenotypic change which a hybridoma culture may undergo is to lose its capacity for growth. This phenomenon is fairly common and occurs quite rapidly when cells expel or fail to replicate a chromosome which encodes a structural protein or enzyme essential for growth. In order to avoid or circumvent some of the undesired characteristics mentioned, frequent and continual re-selection or cloning of hybridoma cultures is essential so that a genetically stable, homogeneous cell line can be maintained.
Hybridoma cultures are cloned using any one of three basic techniques, cloning by limiting dilution, cloning over soft agarose, or cloning by fluorescence activated cell sorting. Cloning by limiting dilution is accomplished by randomly selecting a small group of cells which is then diluted into growth medium at a very low concentration. Small aliquots of the cell suspension, which theoretically contain one or less cells, are then placed into individual growth wells where they may grow into new cultures.
Cloning over soft agarose is accomplished by making very dilute cell suspensions in warmed growth medium containing a suitable amount of agarose. A volume of the cell suspension, which theoretically contains ten to one hundred cells, is plated into a petri dish where it is allowed to cool and solidify. As individual cells grow and divide, colonies of cells result which are held in place by the semi-solid medium. This technique is directly analogous with pour plates in bacteriology.
Lastly, fluorescence activated cell sorting can facilitate the cloning of a hybridoma culture. In this case, laser light is directed at individual cells as they flow through the instrument in a stream of single cells. A light scatter pattern is generated when the dense nuclear material of the cell interferes with the path of the laser beam. Thus, cells can be selected at random based on their ability to scatter laser light. After a cell has been identified, it is deflected away from the stream of cells and is directed into a growth well by means of an automated cell cloning device. The flow method affords a distinct advantage over those previously described in that the fluorescence activated cell sorter is capable of depositing one and only one cell into a particular growth well. The other methods rely on theoretical calculations which statistically make the chances of one cell per well or one cell per unit area likely rather than assured.
All three of the aforementioned techniques rely on the random selection of cells. The successful isolation of a clone with desirable MAb secretion characteristics is no more predictable than the laws of random probability will allow.